Hemostasis is the primary mechanism whereby excessive extravascular blood loss is prevented by the formation of fibrin clots at the site of insult. The physiological mechanism by which these fibrin clots are formed is presently being elucidated. The evidence to date suggests that the clotting mechanism is attributed to a very complex activation cascade of various blood plasma factors each responsible for the sequential activation of the successive zymogen. Proper functioning of this cascade ultimately leads to the formation of fibrin and the cessation of blood loss.
Unfortunately, however, various disease states have been recognized which are attributable to metabolic errors within this blood coagulation cascade either due to a deficiency or total absence of one or more of the blood plasma factors. Chief among these disease states is classic hemophilia or simply hemophilia A. Hemophilia A is a hereditary X chromosome-linked recessive trait which is manifested by an absence of antihemophilic Factor A, which factor is designated by convention as Factor VIII. Therapeutic efforts for treating hemophilia A have focused on supplementing the blood of affected individuals by infusion with concentrates of Factor VIII in various pharmaceutical formulations. Such Factor VIII concentrates have, in the past, largely been prepared by cryoprecipitation of Factor VIII from blood plasma. However, owing to the minute amount of the factor in normal individuals, extremely large quantities of plasma must be processed in order to isolate quantities sufficient for therapy having activity levels adequate for administration in physiologically acceptable volumes of diluent.
With the advent of recombinant DNA technology and the promise of the expression of large quantities of proteins of medical importance (among others), the disadvantages of prior isolation and purification techniques for Factor VIII have become increasingly evident. Indeed, recent publications have reported the successful cloning and expression of the gene encoding Factor VIII (see infra). However, as can be appreciated, even with this technical accomplishment, certain limitations in the efficiency of the overall process may need to be overcome to optimize the benefits flowing from such technology. For example, in the specific case of Factor VIII produced by recombinant DNA techniques, the transformed host cell containing the gene encoding Factor VIII is cultured in a complex medium containing other serum proteins in high concentration. Thus, even after expression of the Factor VIII, recovery and purification of the factor from a culture supernatant containing a heterogeneous population of serum proteins still presents many technical problems. Such difficulties could be alleviated by a means of culturing the recombinant host in the absence of serum proteins in such a manner as to support cell growth without compromising the viability of the host and its ability to express and produce Factor VIII. The present invention is directed to such means and provides for the increased production of recombinant Factor VIII by culturing a host cell carrying a gene capable of directing the expression of Factor VIII in a serum-free nutrient medium supplemented with lipoprotein.